Mycobacterium xenopi: Evidence for Increased Rate of Clinical Isolation.

In light of recent reports of increased isolation of M. xenopi, we reviewed the number of M. xenopi isolates in a hospital setting over five years. A total of 133 isolates from 100 patients were reported, of these isolates, 8 were reported over the first two years, 21 isolates in the third year, 47 isolates in year four and 57 isolates in year five. The specimen sources were mainly respiratory specimens; however a few specimens were isolated from other sources. Clinical data on 12 patients with repeated isolates are presented. Patient conditions upon admission and previous medical histories are shown and compared to earlier reports. An increased awareness of the presence of this organism is necessary since the clinical presentation of patients with M. xenopi can be confused with tuberculosis.


INtrODUctION
There is a growing interest in the diagnosis of potentially pathogenic non-tuberculous Mycobacteria (NTM) (1). This interest was fueled by an increasing number of NTM cases and by the similarity between some of the NTM infections and infections by M. tuberculosis (2,3). Among NTMs of particular interest is Mycobacterium xenopi, which has been a common isolate in Europe and Middle East (4)(5)(6)(7). This organism is becoming increasingly isolated in certain areas of USA (4,8,9). M. xenopi is a slow-growing scotochromogenic Mycobacterium which resembles MAI when tested by biochemical reactions. However it is negative for MAI probe, grows best at 42°C, it does not grow at 25°C (4), and it has a distinct mycolic acid pattern (10).
In this paper, we report a dramatic increase in the number of M. xenopi isolates reported over a five year period, and review the clinical data of patients from whom multiple isolates were obtained.

Data Collection and Analysis
Records of Mycobacterium culture results at Kings County Hospital Center (KCHC) were reviewed for positive M. xenopi cultures during a five year period after 1990. Specimen numbers were listed in Microsoft Works database. Subsequently specimen and patient information were obtained and entered in the same database. Queries were made to obtain the number of isolates and the number of patients with M. xenopi for each month. A Microsoft Works spreadsheet was used to tabulate the data.

Specimen Processing
Non-sterile specimens, such as sputum and bronchial lavage, were digested and decontaminated using NaOH/ NALC (N-Acetyl Cysteine) (11). The final concentrate was suspended in buffer and inoculated into Bactec 12 B medium and/or a Mycobacterium growth indicator tube and a selective 7H11 agar plate. All cultures were incubated at 37°C and screened twice during the first two weeks and once weekly after the second week. When culture was positive, an AFB stain was performed, and the isolate was identified as described below.

Culture Identification
Positive AFB cultures were screened first for M. tuberculosis and Mycobacterium avium-intracellulare (MAI) using Gen-Probe. If both probes were negative, cultures were identified mainly by high performance liquid chromatography (HPLC), based on the method described by Butler et al. (10,12). Briefly, mycolic acids were extracted from 1-2 loopfuls from colonies according to CDC standards, and the final extract of mycolic acids was suspended in 100 µL of methylene chloride and analyzed by HPLC using a C-18 Ultrasphere-X-L analytical column and a mobile phase consisting of a changing gradient. The gradient of methanol and methylene chloride began at 98% and 2% respectively, then changed to 80% and 20% for 1 minute, then changed to 35% and 65% for the following 10 minutes. Chromatograms were analyzed by Piruette software to predict the nearest match from the CDC mycolic acid library. Colonial morphology and growth rate were evaluated for conformation. Chromatogram of M. xenopi isolated in our laboratory (not shown) was similar for isolates identified by HPLC elsewhere (10,12).

Frequency of NTM Isolation
In order to determine if there is an increasing trend in the isolation of NTM, we reviewed positive Mycobacterium cultures over a five year period at KCHC. A total of 12747 specimens were received in year 1, 10500 in year 2 and 8500 in year 5 ( Table 1). The percentages of Mycobacterium tuberculosis (MTB) isolates of all specimens received, for the years one, two and five, were 8.7%, 7.5%, and 5.3%, and the percentages of Mycobacterium avium -intracellulare (MAI) isolates were 6.9%, 10.3%, and 10.6%, respectively. The sharpest increase was observed in NTM group other than MAI, which was 0.9% of all cultures in year one, 1.5% in year two and 3.2% in year five.

review of the Number of M. xenopi cases
The high proportion of M. xenopi isolates in the NTM group prompted us to look further back to track when the number of isolates started to rise and to determine if there is any clinical significance to these isolates. During the five year period, 133 isolates from 100 patients were reported. Table 2 shows the distribution of isolates from these 100 patients in each year. A total of 3 patient were found in year one, 5 patients in year two, 21 patients in year three, 47 patients in year four and 57 patients in year five. 125 M. xenopi isolates were recovered from respiratory sources (sputum, bronchial lavage and bronchial biopsy), 4 isolates from urine, 1 isolate from stool and 3 isolates from body sites that are expected to be sterile.  Bronchial biopsy M. xenopi positive cultures from each specimen source was recorded over a five year period. The total number of all isolates from a particular specimen source is shown (vertical), as well as the total number of isolates for each year (horizontal).

Review of Clinical Data of 12 Patients with Repeated M. xenopi Isolates
In the light of our observation of an increase in M. xenopi isolation, we reviewed the clinical presentation of patients from which repeated isolates of M. xenopi were found. Although M. xenopi was reported in 100 patients over a five year period, only 16 patients were found to have repeated isolates or isolates from normally sterile sites. Among these 16 patients, relevant clinical data for only 12 patients was available for review. As shown in table 3, seven patients were males with a mean age of 50 years (range, 28-76 years) and 5 patients were females with a mean age of 33 years (range, 28-40 years). M. xenopi was isolated from respiratory specimens in 10 patients (cases 1-10), from pericardial fluid in case 11, and from CSF in case 12.

DIscUssION
Our results show an increase in the number of M. xenopi isolates. At our institution, only 33 isolates of M. xenopi were reported during the nine years before 1990 (9). Currently, we report that 3 isolates from 3 patients were found in year 1, 5 isolates from 5 patients in year 2, 21 isolates from 17 patients in year three, 47 isolates from 39 patients in year four, and 57 isolate from 36 patients in year five. Similar observations were also described in Upstate New York (8), New Jersey (4), and Ontario Canada (13).
The clinical relevance of NTM isolates is usually determined by criteria of the American Thoracic Society (ATS) for establishing an NTM disease (14). Since the patients presented in our study had underlying disease(s), it was difficult to determine the clinical relevance of M. xenopi isolation. However, this organism was repeatedly isolated from some of our patients. Therefore, we included patients who were deemed to have clinical infection as judged by repeated isolation of the organism (14).
M. xenopi has recently been isolated from water sources (40,41) and birds are thought to be a natural host reservoir in some countries (42). However, outbreaks of M. xenopi have been associated with colonized hot water sources in private residence and hospitals with the route of infection most likely by the inhalation of aerosols generated during washing or showering (32,41,(43)(44)(45)(46). In our study, most patients presumably acquired the organism outside the hospital since only one patient had a recent prior admission to our hospital. Further epidemiologic studies are necessary to more precisely determine the source and route of infection of these organisms. Although some patients did well on anti-tuberculosis medications, there was no evidence that M. xenopi was the cause of the lung damage, rather than the colonization of an already damaged one.
Since M. xenopi can cause pulmonary symptoms similar to tuberculosis, and the AFB smear may be positive in some cases, mycobacteriology laboratories should be able to identify and report this organism to physicians in the shortest possible time. This is important so that M.xenopi infection can be distinguished from tuberculosis and managed accordingly, in terms of therapy and infection control plans. In our laboratory, the use of HPLC for Mycobacterium identification has substantially reduced the time required for identification of this slow growing organism.